JAK/STAT signaling cascades provide a relative simple and direct connection between ligand binding on the cell surface and gene transcription in the nucleus. These pathways mediate the response to a diverse group of polypeptide ligands which regulate cell growth, differentiation and effector function. One of the best characterized JAK/STAT signaling cascades is the pathway stimulated by interferon- alpha (IFNalpha). Extensive studies of this signal transduction cascade by a number of laboratories has provided a basic understanding of the initial molecular events regulating IFNalpha-induced gene transcription and has established a paradigm for studying other JAK-STAT pathways, as well. During the previous term of this grant, we have explored the molecular interactions between the various components in this cascade: the Tyk2 tyrosine kinase, the IFNaR1 receptor subunit, and the Stat1 and Stat2 transcription factors. In particular, we have: i) identified and characterized the Tyk2 kinase-IFNaR1 receptor complex; ii) identified and characterized the ligand-inducible Phosphotyrosine docking site on IFNaR1 which recruits the SH2 domain of Stat2; and iii) partially reconstituted the IFNalpha signaling pathway using chimeric receptors. Many details of the molecular interactions controlling this pathway, however, remain unknown, including which parts of the Tyk2 kinase are required for the interaction with IFNaR1, how the Stat2-Stat1 heterodimer is released from the receptor, the role of the distal portion of the IFNaR1 subunit and which specific molecules are involved in the negative regulation of this pathway. Four specific aims are proposed to investigate some of the these details: 1. Identify the residues in Tyk2 required for interaction with the IFNaR1 receptor subunit. 2. Define the mechanism of Stat1-Stat2 heterodimer formation. 3. Identify additional IFNaR1-interacting proteins which regulate IFNalpha signaling. 4. Investigate the role of CIS proteins in the regulation of the Tyk2 kinase and/or the IFNaR1 subunit.